The present invention relates to the breeding of a useful plant utilizing a promoter of a plant gene. More specifically, the present invention relates to the breeding of a useful plant by utilizing a promoter gene of catalase gene A (hereinafter referred to as xe2x80x9cCatAxe2x80x9d) from rice.
It is known that an F1 hybrid (first filial generation) which is created through intercultivar crossing may exhibit properties superior to those of its parents, and this has conventionally attracted much attention as a method of breeding crops. As for crops such as rice which experience self-pollination, methods for creating male sterile lines, whose pollen does not have fertility, have been studied as one of the techniques necessary for utilizing this property. Conventionally, plant genetic resource has been searched for male sterile lines, or male sterile lines have been selected by inducing mutagenesis. However, it is not easy to introduce such genes into practical cultivars, and the applications are limited.
Recently, as a method utilizing biotechnology, a method has been proposed in which a promoter that induces expression in anthers and/or pollen is ligated with a gene having a function of inhibiting the formation of such organs (e.g., a gene coding for nuclease, protease, glucanase, etc.) and is introduced into a plant, whereby the formation of fertile pollen is inhibited (for example, Mariani et al., Nature 347:737-741(1990)). Alternatively, methods which involve transcribing an anti-sense RNA of a gene to be expressed at the time of formation of such organs, or introducing a ribozyme which decomposes such mRNAs, by utilizing a promoter which induces expression in anthers and/or pollen, are regarded as promising.
Several kinds of promoters of genes which induce expression in anthers and/or pollen are known for tomato, Arabidopsis thaliana, maize, and the like (for example, Twell et al., Plant Physiol. 91:1270-1274(1989); Paul et al., Plant Molecular Biology 19:611-622(1992): Guerrero et al., Mol. Gen. Genet. 224:161-168(1990)). However, there is a problem in that their activity is too low to put them into practical use. Furthermore, in order to artificially control the formation of anthers and/or pollen, it would be very useful if a promoter which functions in any of the developmental stages of these organs was isolated and characterized, and a cassette containing a highly-active promoter could be created.
Accordingly, it would significantly contribute to the breeding of useful plants, including crops such as rice, if a promoter for anthers and/or pollen which is highly active and permits practical use could be obtained from rice genes.
The present invention relates to the breeding of a plant through genetic engineering techniques, and has an objective of providing an expression cassette for expression in plants and a recombinant plasmid which contain a plant gene promoter having a high activity in anthers and/or pollen, as well as a method for utilizing the same.
The inventors found that the catalase A (CatA) gene from rice exhibits a high promoter activity in anthers and pollen, and accomplished the present invention based on this information.
The present invention provides an expression cassette for expression in plants which allows a heterologous gene to be specifically expressed in anthers and/or pollen. This expression cassette contains a rice CatA gene promoter having a sequence shown as SEQ ID NO: 1, or a sequence which includes a portion thereof and which has a promoter activity equivalent to that of the sequence shown as SEQ ID NO: 1, and a site for inserting a heterologous gene such that the heterologous gene is expressibly linked to the promoter. Herein, the sequence shown as SEQ ID NO: 1 is a sequence including the 5xe2x80x2 upstream region and the first intron of rice CatA structural gene.
The present invention also provides a recombinant plasmid for allowing a heterologous gene to be specifically expressed in anthers and/or pollen. The recombinant plasmid contains a rice CatA gene promoter having a sequence shown as SEQ ID NO: 1, or a sequence which includes a portion thereof and which has a promoter activity equivalent to that of the sequence shown as SEQ ID NO: 1, and a heterologous gene which is expressibly linked to the promoter.
Furthermore, the present invention provides a method for introducing into a plant a heterologous gene which is desired to be specifically expressed in anthers and/or pollen. This method includes the step of transforming a plant cell with the aforementioned recombinant plasmid, and the step of redifferentiating the transformed plant cell to obtain a plant body.
The aforementioned heterologous gene may be a gene which has a function of inhibiting the formation of anthers and/or pollen. By using such a gene in the aforementioned method, a male sterile plant can be created. In the aforementioned method, the plant cell may be either a monocotyledonous cell or a dicotyledonous cell.